Tashiro, Koji*; Kusaka, Katsuhiro*; Hosoya, Takaaki*; Ohara, Takashi; Hanesaka, Makoto*; Yoshizawa, Yoshinori*; Yamamoto, Hiroko*; Niimura, Nobuo*; Tanaka, Ichiro*; Kurihara, Kazuo*; et al.
Macromolecules, 51(11), p.3911 - 3922, 2018/06
Uemura, Takuya*; Kita, Akiko*; Watanabe, Yoshihiko*; Adachi, Motoyasu; Kuroki, Ryota; Morimoto, Yukio*
Biochemical and Biophysical Research Communications, 469(2), p.158 - 163, 2016/01
Tamada, Taro; Shinmi, Daisuke*; Ikeda, Masahiro*; Yonezawa, Yasushi*; Kataoka, Shiro*; Kuroki, Ryota; Mori, Eiji*; Motoki, Kazuhiro*
Scientific Reports (Internet), 5, p.17936_1 - 17936_12, 2015/12
The fully human monoclonal antibody KMTR2 acts as a strong direct agonist for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2), which is capable of inducing apoptotic cell death without cross-linking. To investigate the mechanism of direct agonistic activity induced by KMTR2, the crystal structure of the extracellular region of TRAIL-R2 and a Fab fragment derived from KMTR2 (KMTR2-Fab) was determined to 2.1 resolution. Two KMTR2-Fabs assembled with the complementarity-determining region 2 of the light chain via two-fold crystallographic symmetry, suggesting that the KMTR2-Fab assembly tended to enhance TRAIL-R2 oligomerization. A single mutation at Asn53 to Arg located at the two-fold interface in the KMTR2 resulted in a loss of its apoptotic activity, although it retained its antigen-binding activity. These results indicate that the strong agonistic activity, such as apoptotic signaling and tumor regression, induced by KMTR2 is attributed to TRAIL-R2 superoligomerization induced by the interdimerization of KMTR2.
Tomoyori, Katsuaki; Kurihara, Kazuo; Tamada, Taro; Kuroki, Ryota
JPS Conference Proceedings (Internet), 8, p.036004_1 - 036004_6, 2015/09
We aim to build a high-resolution neutron time-of-flight diffractometer for biomacromolecules at the Materials and Life Science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC) that allows the collection of neutron diffraction data from crystals with unit cells of 250 ;. Considering both the flux and pulse width necessary to realize data collection covering a minimum d-spacing of 2.0 ; and with a unit cell constant of 250 ; we chose a decoupled moderator (DM) as the appropriate source for this high-resolution diffractometer. We considered a simple instrumentation model that includes a moderator, neutron guide, sample size, and neutron detector; we then investigated its spot separation performance and estimated the instrumental parameters for the design of a new diffractometer for protein crystals with large unit cells at J-PARC/MLF. It is preferable to extend the total flight path to resolve Bragg reflections for protein crystals with large unit cells as the scattering angle increases. Meanwhile, to ensure resolvable detection capacity at the middle scattering angle region (2 90), it is necessary to restrict the angular divergence. In the case of 0.2, scattering angles from around 290 to higher backscattering angles are more efficient for protein crystals with large unit cells (250 ) with a resolution of 2.0 .
Yonezawa, Yasushi*; Nagayama, Aiko*; Tokunaga, Hiroko*; Ishibashi, Matsujiro*; Arai, Shigeki; Kuroki, Ryota; Watanabe, Keiichi*; Arakawa, Tsutomu*; Tokunaga, Masao*
Protein Journal, 34(4), p.275 - 283, 2015/08
Nucleoside diphosphate kinase isolated from psychrophilic sp. AS-131 (ASNDK) was expressed in and purified to homogeneity. Comparing to mesophilic NDK isolated from , ASNDK exhibited highly elevated thermolability: (1) expression at 37C as a denatured insoluble form, and (2) 30C lower optimum temperature of enzymatic activity. The subunit structure of ASNDK was suggested to be dimer, as in NDKs isolated from moderate halophiles.
Unno, Masayoshi*; Ishikawa, Kumiko*; Kusaka, Katsuhiro*; Tamada, Taro; Hagiwara, Yoshinori*; Sugishima, Masakazu*; Wada, Kei*; Yamada, Taro*; Tomoyori, Katsuaki; Hosoya, Takaaki*; et al.
Journal of the American Chemical Society, 137(16), p.5452 - 5460, 2015/04
Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IX (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH, a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial "water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 N was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via HO. These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis.
Arai, Shigeki; Yonezawa, Yasushi*; Okazaki, Nobuo*; Matsumoto, Fumiko*; Shibazaki, Chie; Shimizu, Rumi; Yamada, Mitsugu*; Adachi, Motoyasu; Tamada, Taro; Kawamoto, Masahide*; et al.
Acta Crystallographica Section D, 71(3), p.541 - 554, 2015/03
The crystal structure of halophilic -lactamase from sp.560 (HaBLA) was determined using X-ray crystallography. Moreover, the locations of bound Sr and Cs ions were identified by anomalous X-ray diffraction. The location of one Cs specific binding site was identified on HaBLA even in the presence of 9-fold molar excess of Na (90 mM Na /10 mM Cs). This Cs binding site is formed by two main-chain O atoms and an aromatic ring of a side chain of Trp. An aromatic ring of Trp interacts with Cs by the cation- interaction. The observation of a selective and high-affinity Cs binding site provides important information that is useful for designing artificial Cs binding sites useful in bioremediation of radioactive isotopes.
Hiromoto, Takeshi; Honjo, Eijiro*; Noda, Hisanobu*; Tamada, Taro; Kazuma, Kohei*; Suzuki, Masahiko*; Blaber, M.; Kuroki, Ryota
Protein Science, 24(3), p.395 - 407, 2015/03
UDP-glucose: anthocyanidin 3--glucosyltransferase (UGT78K6) from catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 , 2.55 , 2.70 and 1.75 respectively. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180 rotation about the O1-O3 axis of the flavonoid backbones observed in GT1 and UGT78G1. These substrate recognition schemes suggest the potential for controlled synthesis of natural pigments.
Kuroki, Ryota; Miyazaki, Hiroshi*; Kato, Takashi
Ketsueki Furonteia, 25(2), p.171 - 180, 2015/02
no abstracts in English
Tashiro, Koji*; Hanesaka, Makoto*; Yamamoto, Hiroko*; Wasanasuk, K.*; Jayaratri, P.*; Yoshizawa, Yoshinori*; Tanaka, Ichiro*; Niimura, Nobuo*; Kusaka, Katsuhiro*; Hosoya, Takaaki*; et al.
Kobunshi Rombunshu, 71(11), p.508 - 526, 2014/11
The crystal structure analysis of various polymer substances has been reviewed on the basis of wide-angle high-energy X-ray and neutron diffraction data. The progress in structural analytical techniques of polymer crystals have been reviewed at first. The structural models proposed so far were reinvestigated and new models have been proposed for various kinds of polymer crystals including polyethylene, poly(vinyl alcohol), poly(lactic acid) and its stereocomplex etc. The hydrogen atomic positions were also clarified by the quantitative analysis of wide-angle neutron diffraction data, from which the physical properties of polymer crystals have been evaluated theoretically. The bonded electron density distribution has been estimated for a polydiacetylene single crystal on the basis of the so-called X-N method or by the combination of structural information derived from X-ray and neutron diffraction data analysis. Some comments have been added about future developments in the field of structure-property relationship determination.
Adachi, Motoyasu; Hirayama, Hiroshi; Shimizu, Rumi; Sato, Katsuya; Narumi, Issey*; Kuroki, Ryota
Protein Science, 23(10), p.1349 - 1358, 2014/10
Pleiotropic protein promoting DNA repair A (PprA) is a key protein that facilitates the extreme radioresistance of . To clarify the role of PprA in the radioresistance mechanism, the interaction between recombinant PprA expressed in Escherichia coli with several double-stranded DNAs was investigated. In a gel-shift assay, the band shift of supercoiled pUC19 DNA caused by the binding of PprA showed a bimodal distribution, which was promoted by the addition of 1 mM Mg, Ca, or Sr ions. The dissociation constant of the PprA-supercoiled pUC19 DNA complex, calculated from the relative portions of shifted bands, was 0.6 M with a Hill coefficient of 3.3 in the presence of 1 mM Mg acetate. This indicates that at least 281 PprA molecules are required to saturate a supercoiled pUC19 DNA, which is consistent with the number of bound PprA molecules estimated by the UV absorption of the PprA-pUC19 complex purified by gel filtration. This saturation also suggests linear polymerization of PprA along the dsDNA. On the other hand, the bands of linear dsDNA and nicked circular dsDNA that eventually formed PprA complexes did not saturate, but created larger molecular complexes when the PprA concentration was greater than 1.3 M. This result implies that DNA-bound PprA aids association of the termini of damaged DNAs, which is regulated by the concentration of PprA.
Shinka O Tsuzukeru Kozo Seibutsugaku; Aratana Tampakushitsu Kino No Kaimei To Soshutsu, p.93 - 109, 2014/09
no abstracts in English
Nihon No Kesshogaku, 2; Sono Kagayakashii Hatten, P. 392, 2014/07
no abstracts in English
Arai, Shigeki; Yonezawa, Yasushi*; Ishibashi, Matsujiro*; Matsumoto, Fumiko*; Adachi, Motoyasu; Tamada, Taro; Tokunaga, Hiroko*; Blaber, M.; Tokunaga, Masao*; Kuroki, Ryota
Acta Crystallographica Section D, 70(3), p.811 - 820, 2014/03
In order to clarify the structural basis of halophilic characteristics of an alkaline phosphatase derived from the moderate halophile sp.593 (HaAP), the tertiary structure of HaAP was determined to 2.1 resolution by X-ray crystallography. Structural properties of surface negative charge and core hydrophobicity are shown to be intermediate between halophile and non-halophile characteristics, and may explain the unique functional adaptation to a wide-range of salt concentration.
Adachi, Motoyasu; Arai, Shigeki; Hiromoto, Takeshi; Kuroki, Ryota
Hamon, 24(1), p.45 - 49, 2014/02
Protein structure analysis using neutron diffraction (neutron protein crystallography; NPC) is gaining greater importance in the understanding of structure and function relationships of biological macromolecules such as proteins and DNA. Current developments of neutron diffractometers installed at the JAEA research reactor and pulsed neutron source permit observation of the locations of hydrogen atoms and hydrating water molecules and help understanding of important mechanisms of chemical reactions catalyzed by biological macromolecules. Here, we introduce practical approaches of NPC including sample preparation, crystal growth, structure determination and utilization of information obtained from NPC.
Murakawa, Takeshi*; Hayashi, Hideyuki*; Sunami, Tomoko; Kurihara, Kazuo; Tamada, Taro; Kuroki, Ryota; Suzuki, Mamoru*; Tanizawa, Katsuyuki*; Okajima, Toshihide*
Acta Crystallographica Section D, 69(12), p.2483 - 2494, 2013/12
The crystal structure of a Cu amine oxidase from was determined at 1.08 resolution with the use of low-molecular-weight polyethylene glycol (LMW PEG; average molecular weight 200) as a cryoprotectant. The final crystallographic -factor and value are 13.0% and 15.0%, respectively. Several molecules of LMW PEG were found to occupy cavities in the protein interior including the active site, which resulted in the marked reduction of the overall factor and consequently led to a sub-atomic resolution structure for a relatively large protein with a monomer molecular weight of 70,000. About 40% of all the presumed hydrogen atoms were observed as clear electron densities in the - difference map. Multiple minor conformers were also identified for many residues. Anisotropic displacement fluctuations were evaluated in the active site that contains a post-translationally derived quinone cofactor and a Cu atom. Furthermore, diatomic molecules, most likely molecular oxygen, are bound to the protein, one of which is located in the region that has been previously proposed as an entry route for the substrate dioxygen from the central cavity of the dimer interface to the active site.
Yamada, Mitsugu*; Tamada, Taro; Takeda, Kazuki*; Matsumoto, Fumiko*; Ono, Hiraku*; Kosugi, Masayuki*; Takaba, Kiyofumi*; Shoyama, Yoshinari*; Kimura, Shigenobu*; Kuroki, Ryota; et al.
Journal of Molecular Biology, 425(22), p.4295 - 4306, 2013/11
NADH-Cytochrome reductase (b5R), a flavoprotein consisting of NADH and flavin adenine dinucleotide (FAD) binding domains, catalyzes electron transfer from the two-electron carrier NADH to the one-electron carrier cytochrome (Cb5). The crystal structures of both the fully reduced form and the oxidized form of porcine liver b5R were determined. In the reduced b5R structure determined at 1.68 resolution, the relative configuration of the two domains was slightly shifted in comparison with that of the oxidized form. This shift resulted in an increase in the solvent-accessible surface area of FAD and created a new hydrogen-bonding interaction between the N5 atom of the isoalloxazine ring of FAD and the hydroxyl oxygen atom of Thr66, which is considered to be a key residue in the release of a proton from the N5 atom. The isoalloxazine ring of FAD in the reduced form is flat as in the oxidized form and stacked together with the nicotinamide ring of NAD. Determination of the oxidized b5R structure, including the hydrogen atoms, determined at 0.78 resolution revealed the details of a hydrogen-bonding network from the N5 atom of FAD to His49 via Thr66. Both of the reduced and oxidized b5R structures explain how backflow in this catalytic cycle is prevented and the transfer of electrons to one-electron acceptors such as Cb5 is accelerated. Furthermore, crystallographic analysis by the cryo-trapping method suggests that re-oxidation follows a two-step mechanism. These results provide structural insights into the catalytic cycle of b5R.
Adachi, Motoyasu; Shimizu, Rumi; Kuroki, Ryota; Blaber, M.
Journal of Synchrotron Radiation, 20(6), p.953 - 957, 2013/11
Symfoil-4P is a protein exhibiting the threefold symmetrical beta-trefoil fold designed based on the human acidic fibroblast growth factor. First three asparagine-glycine sequences of Symfoil-4P are replaced with glutamine-glycine (Symfoil-QG) or serine-glycine (Symfoil-SG) sequences protecting from deamidation, and His-Symfoil-II was prepared by introducing a protease digestion site into Symfoil-QG so that Symfoil-II has three complete repeats after removal of the N-terminal histidine tag. The Symfoil-QG and SG and His-Symfoil-II proteins were expressed in as soluble protein, and purified by nickel affinity chromatography. Symfoil-II was further purified by anion-exchange chromatography after removing the HisTag by proteolysis. Symfoil-QG and II crystals gave 1.5 and 1.1, resolution, respectively. The refined crystal structure of Symfoil-II showed pseudo-threefold symmetry as expected from other Symfoils.
Hiromoto, Takeshi; Honjo, Eijiro*; Tamada, Taro; Noda, Hisanobu*; Kazuma, Kohei*; Suzuki, Masahiko*; Kuroki, Ryota
Journal of Synchrotron Radiation, 20(6), p.894 - 898, 2013/11
Flowers of the butterfly pea () accumulate a group of polyacylated anthocyanins, named ternatins, in their petals. The first step in ternatin biosynthesis is the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin, a reaction catalyzed in by UDP-glucose:anthocyanidin 3--glucosyltransferase (3GT-A; AB185904). To elucidate the structure-function relationship of 3GT-A, recombinant 3GT-A was expressed in and its tertiary structure was determined to 1.85 , resolution by using X-ray crystallography. The structure of 3GT-A shows a common folding topology, the GT-B fold, comprised of two Rossmann-like // domains and a cleft located between the N- and C-domains containing two cavities that are used as binding sites for the donor (UDP-Glc) and acceptor substrates. By comparing the structure of 3GT-A with that of the flavonoid glycosyltransferase GT1 from red grape () in complex with UDP-2-deoxy-2-fluoro glucose and kaempferol, locations of the catalytic His-Asp dyad and the residues involved in recognizing UDP-2-deoxy-2-fluoro glucose were essentially identical in 3GT-A, but certain residues of GT1 involved in binding kaempferol were found to be substituted in 3GT-A. These findings are important for understanding the differentiation of acceptor-substrate recognition in these two enzymes.
Yamada, Taro*; Kurihara, Kazuo; Onishi, Yuki*; Tamada, Taro; Tomoyori, Katsuaki; Masumi, Kenji*; Tanaka, Ichiro*; Kuroki, Ryota; Niimura, Nobuo*
Biochimica et Biophysica Acta; Proteins and Proteomics, 1834(8), p.1532 - 1538, 2013/08
The protonation states and hydration structures of the -thrombin-bivalirubin complex were studied by joint XN refinement of the single crystal X-ray and neutron diffraction data at resolutions of 1.6 and 2.8 , respectively. The atomic distances were estimated by carrying out X-ray crystallographic analysis at 1.25 resolution. The complex represents a model of the enzyme-product (EP) complex of -thrombin. The neutron scattering length maps around the active site suggest that the side chain of H57/H was deuterated. The joint XN refinement showed that occupancies for D1 and D2 of H57/H were 1.0 and 0.7, respectively. However, no significant neutron scattering length density was observed around the hydroxyl oxygen O of S195/H, which was close to the carboxylic carbon atom of dFPR-COOH. These observations suggest that the O atom of S195/H is deprotonated and maintains its nucleophilicity in the EP complex. In addition to the active site, the hydration structures of the S1 subsite and the Exosite I, which are involved in the recognition of bivalirudin, are presented.